Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Abstract Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae , and the eukaryotic microalgae Phaeodactylum tricornutum . We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 10 5 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae , and P. tricornutum , with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans .